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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10.01.1996-13.02. 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP compliant study undertaken to a standard considered to be equivalent to international test guidelines for a chromosome aberration study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996
Reference Type:
publication
Title:
Di(isononyl) phthalate (DINP) and di(isodecyl) phthalate (DIDP) are not mutagenic.
Author:
McKee RH, Przygoda RT, Chirdon MA, Engelhardt G, Stanley M.
Year:
2000
Bibliographic source:
J Appl Toxicol. 2000 Nov-Dec;20(6):491-7.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich
EC Number:
271-090-9
EC Name:
1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich
Cas Number:
68515-48-0
IUPAC Name:
bis(7-methyloctyl) phthalate
Constituent 2
Reference substance name:
DINP
IUPAC Name:
DINP
Details on test material:
- Name of test material (as cited in study report): Di-isononyl phthalate
- Substance type: Technical material
- Physical state: Colourless liquid
-Stability under test conditions: Room temperature

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
WBL clone, supplied by Merck Research Laboratories, West Point , Pennsylvania, USA. Received 5 June 1992
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat
Test concentrations with justification for top dose:
0 (vehicle control), 5, 10, 20, 40, 80 and 160 micrograms/ml. The treatment groups and test concentrations are tabulated in the 'overall remarks and attachments' window below.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Solubility
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
None

Migrated to IUCLID6: and, N-Methyl-N-Nitro-N-Nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period: None
- Exposure duration: 3 or 20 hours
- Expression time (cells in growth medium): 20 or 44 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 or 44 hours



SPINDLE INHIBITOR (cytogenetic assays): Yes (Colcemid)
STAIN (for cytogenetic assays): Yes (5% Giemsa)


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 200 per per treatment group


DETERMINATION OF CYTOTOXICITY:
- Method: mitotic index; number of mitotic cells per 1000 total cell count


OTHER EXAMINATIONS:
- Determination of polyploidy: Included in aberrration totals. If marked increases had been noted the frequency would have been calculated based on 500 metaphases in the particular flask
- Determination of endoreplication: Not included in aberration totals. If marked increase had been noted the frequency would have been calculated based on 500 metaphases in the particular flask.



Evaluation criteria:
See 'overall remarks and attachments' window below
Statistics:
See 'overall remarks and attachments' window below

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data (unlikely as substance has low vapor pressure and a Log P o/w relatively high)
- Water solubility: Doses were significantly higher than the estimated water solubility of DINP (2 µg/ml).
- Precipitation: precipitation was thought to have occured on some occasions at 40, 80 or 160 µg/ml
- Other confounding effects: No data


RANGE-FINDING/SCREENING STUDIES: YES

A toxicity pre-test was performed to determine appropriate doses for the main study.


COMPARISON WITH HISTORICAL CONTROL DATA: No quantitative data reported.






Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

A tabulated summary of results is presented in the 'overall remarks and attachments' window below.

No statistically significant differences were observed between the test and vehicle control groups in the percentage of aberrant cells following treatment with the test substance at 40, 80 or 160 µg/l, either with or without metabolic activation, for the initial or repeat asssays.

Although there was a statistically significant trend of the series without metabolic activation for the 20-hour harvest in the repeat asssay, the response at the high dose (160 µg/ml) was not extreme and is within the normal range of the vehicle control. It was not supported by an observable dose response, and was not supported by other results at other harvests. Examination of the replicates showed that one flask at 160 µg/ml, had 6 aberrant cells out of 100 while the other flask at the same dose had only 2 aberrant cells out of 100. It appeared that the one flask (6/100) was anomalous and not representative of the other data. Based on this data analysis, there was no statistically significant relationship between dose and the number of aberrant cells.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance did not induce chromosomal aberrations in chinese hamster ovary cells
Executive summary:

Chinese hamster ovary (CHO) cells were exposed to di-isononyl phthalate (DINP) in acetone at concentrations of 5,10, 20, 40, 80 or 100 µg/ml media, for 3 hours (with and without metabolic activation) in an initial assay, or 3 hours (with metabolic activation) and 20 hours (without metabolic activation) in a repeat assay, and harvested after 20 or 44 hours incubation, respectively. DINP did not induce chromosomal aberrations in the cultured CHO cells at any of the evaluted dose levels (40, 80 and 160 µg/ml) with and without metabolic activation.

The study was considered acceptable for classification, and satisfied the requirements for mammalian cell chromosomal aberration studies.